Mosivand, Z. (2015) Molecular detection of Pseudomonas aeroginosa by Gold nanoparticles probe. Masters thesis, University of Zabol.
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Abstract
Molecular techniques, such as polymerase chain reaction (PCR) are rapid and reliable diagnostic methods for detection of microbial pathogens. In recent decades, the Pseudomonas aeruginosa antibiotic treatment, has been identified as one of the most important nosocomial pathogens that causes acute and chronic infections in patients admitted to the hospital. Many PCR based diagnostic methods for detection of Pseudomonas aeruginosa have been established, however many of these protocols detect only one target gene and do not provide a perfect and reliable detection method to identify the bacteria, because Pseudomonas aeruginosa species show high genotypic diversity. To overcome these problems, several PCR methods have been used. Meanwhile due to genetic exchanges between Pseudomonas aeruginosa and closely bacteria the specificity of this technique is often low, therefore there is a need to design a highly specific technique based on specific probes and Multiplex PCRs to detect Pseudomonas aeruginosa. In this study, two molecular methods used were Multiplex PCR and gold nanoparticles colorimetric assay then the sensitivity and specificity of the test was evaluated. Optimization of Multiplex PCR and its sensitivity and specificity was performed using four gene-specific Pseudomonas aeruginosa (oprL, oprI, toxA and 16s rDNA) and results showed 100% specificity. Sensitivity of this method to detect bacteria using colony counting was 80 CFU/mL; in addition the sensitivity based on genomic DNA concentration was 0.05ng/μL. Eventually confirmation of detection was investigated by gold nanoparticle probes. Probe attached to gold nanoparticles in the presence of the target DNA caused aggregation of gold nanoparticles in the form of connected network and resulted in colour exchange. The colour change indicates the presence of the target molecule in the sample and could be observed by naked eye. In this study etiolated probe was designed based on 16s rDNA gene sequence and after hybridization of probes, fluctuations in wavelength between 400 and 600 nm was investigated. Results showed that the wavelength of gold nanoparticles was shifted from 524 to 558 nm. This method indicated 100% specificity and colorimetric detection sensitivity based on the concentration of genomic DNA achieved at 0.01ng / μL, which was about 5 times higher than PCR using 16s rDNA gene.
| Item Type: | Thesis (Masters) |
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| Uncontrolled Keywords: | Pseudomonas aeruginosa, gold nanoparticle probes, colorimetric assay, Multiplex PCR, oprL ،oprI ،toxA and 16s rDNA genes |
| Subjects: | Q Science > QH Natural history > QH301 Biology |
| Depositing User: | admin admin1 admin2 |
| Date Deposited: | 24 Oct 2016 08:59 |
| Last Modified: | 24 Oct 2016 08:59 |
| URI: | http://eprints.uoz.ac.ir/id/eprint/886 |
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